The present invention relates generally to treatment of patients with leukemia. In particular, the present invention relates to the apoptosis of leukaemic cells.
The ubiquitous, amiloride-sensitive, growth factor activatable sodium/hydrogen exchanger isoform 1 (NHE-1) represents one of the primary mechanisms by which cells regulate intracellular pH (pHi) (1, 2). The human NHE-1 is an 815 amino acid membrane protein transporter produced from the 70 kb long APNH gene on chromosome 1 (3). The protein has 12 membrane spanning segments containing the amiloride-binding site and the highly conserved Na+/H+ binding site. The cytoplasmic domain contains the pH sensor and maintenance sites (2). It also contains regions which activate the antiporter when growth factors, mitogens or non-mitogenic signals act on the cell (2). Activation of NHE-1 results in a 1:1 efflux of H+ and influx of Na+ ions, with a concomitant increase in pHi. Increased pHi is associated with cell stimulation and proliferation (1). Using the interleukin-3 (IL-3)-dependent stem cell line, FDCP-mix, Whetton et al (4) demonstrated that IL-3 activated the NHE and that the resulting intracellular alkalinization was a signal for proliferation of these cells. When granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to stimulate macrophage proliferation, activation of the NHE was detected (5). Removal of growth factors such as IL-2 from IL-2 dependent cytotoxic T cells resulted in a decrease in pHi and the onset of apoptosis (6, 7). We have demonstrated that murine bone marrow cells could be stimulated by interaction of the xcex14 integrin subunit with fibronectin, in the absence of growth factors, causing activation of NHE-1, an increase in pHi and increased colony formation of hemopoietic stem and progenitor cells (8). Several reports using different leukemic cell lines demonstrated that if the Na+/H+ exchanger was inhibited by amiloride analogues, acidification of the cells occurred with induction of apoptosis (9-11). We hypothesize that cells maintaining a high rate of proliferation should exhibit a sustained increase in pHi relative to normal cells as a result of activation of the sodium/hydrogen exchanger. Here we show that leukemia cell lines and primary patient leukemic samples exhibit a greater pHi than normal cells, that pHi is correlated with cell cycle status and that inhibition of NHE-1 in leukemic patient samples results in a decrease in pHi and increase in apoptosis. We therefore demonstrate that an inhibitor of NHE-1 has anti-leukemic activity.
A method is provided to sort cells by flow cytometry into subpopulations of proliferating and non-proliferating cells and to induce apoptosis in proliferating hemopoietic and leukemic cells by inhibiting the Na+/H+ exchanger, thereby lowering the internal pH.